EP1369126A1 - Utilisation de peptides de régions de microsatellites codantes et mutantes de déphasage pour traiter le cancer - Google Patents

Utilisation de peptides de régions de microsatellites codantes et mutantes de déphasage pour traiter le cancer Download PDF

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Publication number
EP1369126A1
EP1369126A1 EP02008774A EP02008774A EP1369126A1 EP 1369126 A1 EP1369126 A1 EP 1369126A1 EP 02008774 A EP02008774 A EP 02008774A EP 02008774 A EP02008774 A EP 02008774A EP 1369126 A1 EP1369126 A1 EP 1369126A1
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Prior art keywords
frameshift
gene
peptides
tract
polypeptides
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EP02008774A
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German (de)
English (en)
Inventor
Magnus Von Knebel Doeberitz
Johannes Gebert
Michael Linnebacher
Stefan Wörner
Rüdiger Ridder
Peer Bork
Y.R Yuan
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Roche MTM Laboratories AG
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Roche MTM Laboratories AG
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Priority to EP02008774A priority Critical patent/EP1369126A1/fr
Priority to PCT/EP2003/004083 priority patent/WO2003087162A2/fr
Priority to AU2003232485A priority patent/AU2003232485A1/en
Priority to US10/511,698 priority patent/US8053552B2/en
Publication of EP1369126A1 publication Critical patent/EP1369126A1/fr
Priority to US13/284,660 priority patent/US8821864B2/en
Priority to US14/340,467 priority patent/US20140363831A1/en
Priority to US16/120,981 priority patent/US20190265244A1/en
Priority to US17/302,694 priority patent/US20220091122A1/en
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination

Definitions

  • the present invention relates to a method for the treatment of cancer, especially of DNA mismatch repair deficient (MMR) sporadic tumours and HNPCC associated tumours, which uses immuno therapy with combinatorial mixtures of tumour specific frameshift peptides to elicit a cytotoxic T-cell response specifically directed against tumour cells.
  • the method may be used for prevention of cancers and for the curative treatment of cancers and precancers.
  • CIN chromosomal instability
  • MSI microsatellite instability
  • MSI occurs in about 90% of hereditary nonpolyposis colorectal cancers (HNPCC) as well as in about 15% of sporadic tumours of the colon and other organs, and is caused by mutational inactivation of different DNA mismatch repair genes.
  • HNPCC hereditary nonpolyposis colorectal cancers
  • mutant proteins or peptides encoded by expressed mutant genes may elicit a specific cellular immune response and thus may be recognized by CTL. This is especially true concerning mutations resulting from chromosomal instabilities, but also pertains to more subtle genetic alterations, such as small deletions and insertions in microsatellites.
  • the cancer cells are characterized by the expression of neo-peptides, which arose from the genetic alterations. These proteins are not present in normal, non cancerous tissues. Thus the neo-peptides may be used to distinguish on a molecular level between tumours and normal tissues. As tools suitable for the molecular discrimination antigen specific molecules or cells may be used. Thus it is possible to elicit an immune response against frameshift peptides, as might arise from MSI mutations, specific for tumours. Using cytotoxic T lymphocytes or FSP-specific antibodies it is thus possible to specifically attack and eliminate tumour cells in organisms and tissues.
  • the T-cell mediated immune response provides a strong and specific selection pressure for the rapidly growing tumour cells (Tomlinson I, et al., Nat. Med. 1999; 5 : 11-2).
  • tumour evolution is forced to circumvent the immune surveillance by the cellular immune response.
  • mutations in genes of the antigen processing or presenting machinery arise in MMR-deficient tumour cells.
  • evasion of the immune surveillance by acquiring ⁇ 2-microglobulin mutations has been observed at high frequency in MSI + tumour cells (Bicknell DC, et al., Curr. Biol 1996; 6 : 1695-7).
  • Other targets of specific mutation or down-regulation of expression are TAP1/TAP2 or HLA alleles.
  • the promising therapies based on immune response directed against frameshift peptides in tumour tissues are prone to fail in a not yet determined number of cases of tumours.
  • the approaches for the use of the vaccination therapy of cancer mediated by frameshift peptides might be limited by the fact, that the potential immunogenic frameshift peptides are not always detectable by the immune system due to mutations in the antigen presenting and processing properties of the cancer cells.
  • the present invention provides methods for enhanced therapeutic methods based on immune response directed against frameshift peptides in tumour cells.
  • the present invention is based on the inventors findings, that a mixture of frameshift peptides chosen according to combinatorial parameters minimizes the escape of particular populations of tumour cells from immunogenic elimination and may be used as a vaccine against a wide variety of DNA mismatch repair deficient tumours.
  • the invention is based on the fact, that the immune escape of tumour cells is directed towards single immunogenic epitopes (frameshift peptides) in a potentially restricted subpopulation of the tumour cells. In the vast majority of MSI+ tumours various mutations may be found within the genome of the affected cells. So a combinatorial vaccination approach including several antigenic peptides could overcome this obstacle.
  • Another aspect of the present invention is a method for vaccination against MSI + tumours using said set of frameshift peptides.
  • a third aspect of the present invention is a method for treatment of MSI + tumours using a set of frameshift peptides, that elicit an immune response directed against a wide range of tumours.
  • Frameshift polypeptides as used herein shall comprise any polypeptides or fragments thereof, that arise by a frameshift mutation within a coding microsatellite sequence of a gene.
  • a gene may harbour one or more coding microsatellite regions, that may give rise to frameshift peptides.
  • the coding microsatellites according to the present invention comprise mononucleotide repeats, dinucleotide repeats, trinucleotide repeats, tetranucleotide repeats and pentanucleotide repeats of any length.
  • coding microsatellites contain preferably at least 3 and more preferably at least 5 repeats of the respective nucleotide pattern (1-5 nucleotides, which are repeated).
  • the frameshift polypeptides as used according to the present invention comprise at least 1, more preferred at least 2 and even more preferred at least 3 amino acids of the mutated part of the polypeptide. Additionally, the frameshift polypeptides may comprise fragments of the originally non-mutated proteins and/or may be fused to any other polypeptide sequences suitable for the purposes of the present invention. Examples of such polypeptide sequences are linker sequences, or structural peptide sequences, such as beta barrels, loop sequences etc. that facilitate the immunogenicity of the frameshift sequences according to the present invention within the fusion polypeptide.
  • Frameshift polypeptides suitable for the method disclosed herein have to be immunogenic polypeptides. This requires, that the polypeptides may stimulate immune responses in host organisms either in the form the polypeptides adopt in their natural environment and/or especially in the form the polypeptides adopt after processing by the cellular antigen processing and presenting machinery.
  • the frameshift polypeptides may also be represented by nucleic acids coding for said polypeptides. These nucleic acids may for example be used for the in situ expression of the respective polypeptides. For the purpose of expression of nucleic acids, the particular nucleic acids may be joined with suitable other nucleic acid sequences, that enable for the cloning and expression of said nucleic acids encoding the frameshift polypeptides.
  • Mutation as used in the context of the present invention may comprise insertions or deletions of one or several nucleotides (or nucleotide repeats) within the specified microsatellite sequences.
  • the number of nucleotides to be inserted or deleted is not 3 or must not be divisible by 3, such that the mutation leads to a frameshift with respect to the translational reading frame of the downstream nucleic acid sequence.
  • the nucleic acid sequence downstream of the mutation point will render a polypeptide-sequence different from the native sequence encoded by the respective gene.
  • the mutation in these cases leads to a novel peptide sequence (a neo-peptide).
  • the new peptide sequence is short due to the fact, that novel stop codons arise from the shift in the reading frame.
  • the method according to the present invention may be used for the treatment of disorders associated with frameshift mutations within coding microsatellite regions of genes.
  • disorders comprise for example degenerative diseases, such as neurodegenerative diseases, vascular disorders, disorders caused by stress, such as oxidative stress, chemically induced stress, irradiation induced stress, etc. and cancers including all sporadic cancers as well as HNPCC associated cancers.
  • Cancers as used herein may comprise e.g. colorectal cancer, small cell lung cancer, liver cancer (primary and secondary), renal cancer, melanoma, cancer of the brain, head and neck cancer, gastrointestinal cancers, leukemias, lymphomas. prostate cancer, breast cancer, ovary cancer, lung cancer, bladder cancer etc..
  • the vaccines according to the present invention comprise one or more sets of frameshift polypeptides.
  • a set of frameshift polypeptides used according to the method disclosed herein comprises at least 5, more preferably at least 7 and most preferred at least 10 frameshift polypeptides. Due to immunological as well as practical concerns the set of frameshift peptides used as a vaccine may not include an unlimited number of frameshift peptides. Preferably the set of frameshift peptides comprises at maximum 15, in a more preferred embodiment at maximum 20 and in the most preferred embodiment of the invention at maximum 30 frameshift polypeptides. In order to ensure a maximum range of disorders to be addressed by the selected set of frameshift polypeptides the members of the set have to be selected by reasonable considerations.
  • the frameshift polypeptides to be used as a set according to the present invention are selected with respect to a number of parameters characterizing said polypeptides.
  • Mutation frequency as used in the context of the present invention pertains to the percentage of samples within a defined range of total samples, which show a particular mutation. Any method suitable for the determination of the percentage of individuals in a range of samples displaying the existence of a particular genotype or phenotype (with respect to the expression of polypeptides) may be employed for the determination of the frequency according to the present invention.
  • the frequencies may be determined e.g. as described below in example 1.
  • the frequency may be the frequency of frameshift mutation within a coding microsatellite region or a frequency of expression of a frameshift polypeptide. Furthermore the frequency may be e.g. a frequency over a total of different tumour entities, the frequency with respect to a particular tumour entity, a frequency over several tumours entities restricted to particular stages of tumourigenesis or a frequency with respect to a particular tumour entity restricted to particular stages of tumourigenesis.
  • the frequency according to the present invention has to be determined taking into account a range of samples sufficient to render significant data. For example preferably at least 50 to 100 tumours may be included in the range of samples analyzed. In case, that a smaller number of samples has been taken into account for determination of the frequency, a variation of the determined frequency may take place, if the range of samples is broadened.
  • any frequency as determined may be used as a tool for the choice of a set of frameshift polypeptides according to the present invention.
  • a largest possible number of samples has to be taken into account.
  • a frequency may also be determined with respect to a restricted number of samples. This may especially be true, if the data for the restricted population of samples indicates a quite high frequency of a particular peptide, and thus implies a high therapeutic value for the respective peptide.
  • the population of samples may be restricted.
  • the mutation may occur at any time during the tumour evolution and may be persistent or may be eliminated from the genome.
  • the frequency may comprise the frequency of the expression of a peptide at a particular stage of tumourigenesis or the occurrence of a genetic mutation at a particular defined stage of tumorigenesis.
  • the frequency for the mutation is determined taking into account the widest possible range of samples.
  • the method disclosed herein may be especially useful for the preventive vaccination of tumours.
  • the frequencies of mutation are related to specified tumour entities.
  • the method according to the present invention may e.g. especially be useful for immuno-therapeutic approaches in treatment of tumours or in the preventive vaccination of particular subpopultions with an elevated risk for the occurrence of particular tumours.
  • the frequency may be related to particular stages in tumourigenesis of particular tumours.
  • This embodiment may be e.g. especially useful for the treatment of diagnostically defined tumours or for adjuvant treatment of tumours simultaneously or following primary treatment of tumours.
  • frameshift polypeptides that occur with a high frequency, the probability, that a particular tumour expresses the peptide and may thus be recognized by antibodies or antigen-recognizing cells, increases. By combination of multiple such polypeptides, the probability to address particular tumour cells further is raised.
  • Preferred frameshift polypeptides according to the present invention occur in at least 25%, more preferred frameshift polypeptides in at least 30% and most preferred frameshift polypeptides in at least 33% of the cases of the particular condition to be treated by the method according to the present invention.
  • the polypeptides are chosen to be expressed with a high frequency, thus the set of polypeptides may be limited to a number of members and nonetheless may cover a range of occurring diseases as wide as possible. For example a set of 10 polypeptides, each occurring with a frequency of more than 30 percent will statistically cover a range of more than 95% of the potentially existing disorders, as far as they have been included in the studies leading to the respective frequencies. Using 7 polypeptides requires the application of polypeptides with higher frequencies in order to cover a range of about 95% of potentially existing tumours.
  • the set of polypeptides comprises in a preferred embodiment at least 5, in a more preferred embodiment at least 7 and in the most preferred embodiment at least 10 different frameshift polypetides.
  • a second aspect influencing the choice of a suitable set of frameshift polypeptides concerns the type of mutation found in the microsatellite region.
  • Frameshift mutations microsatellites are usually due to DNA polymerase slippage and may be characterized by the type of the repeat. Thus in mono nucleotide repeats this type of mutation renders 1 nt insertion or deletion.
  • dinucleotide repeats and tetranucleotide repeats mutations are insertions or deletions of 2 or 4 nt respectively (Polypeptides encoded by genes with coding microsatellites and the respective polypeptides encoded by genes with frameshift mutations are given in Figure 5).
  • (-1) mutations occur in mononucleotide repeats (MNRs).
  • one nucleotide is deleted such that the reading frame is shifted by one nucleotide toward the 5' end of the gene compared to the original reading frame.
  • This type of mutation renders a reading frame identical to that produced by (+2) mutations, which arise from two nucleotide insertions in the respective microsatellites.
  • the respective (-1) or (+2) polypeptides might differ by one amino acid.
  • the other frameshift mutation variant leading to frameshift mutations differing from (-1) mutations concerning the resulting reading frame is the (+1) mutation, arising from one nucleotide insertion, thus rendering a reading frame shifted one nucleotide toward the 3' end of the gene compared to the original reading frame.
  • This mutation type gives a reading frame identical to that of (-2) mutations, wherein the encoded polypeptides differ by one amino acid.
  • (-3) and (+3) mutations are irrelevant according to the present invention, for they do not give rise to frameshift polypeptides.
  • the polypeptides included within the set shall be selected as to cover the widest possible range of tumours.
  • a set comprises for example (-1 ) frameshift polypeptides and additionally (+1) frameshift polypeptides.
  • a further aspect influencing the choice of the member polypeptides included in a set of frameshift polypeptides according to the present invention is the involvement of the gene encoded for by the coding sequence containing the respective microsatellite in particular biochemical pathways.
  • biochemical pathway is used with a rather broad meaning herein.
  • Biochemical pathways as used within this document shall for example include signal transduction pathways, enzymatic pathways, metabolic pathways, the apoptosis pathway, DNA repair or polymerization pathways, the pathway of meiosis etc..
  • members of different pathways are included in the set.
  • At least 5 different pathways in a more preferred embodiment at least 4 different pathways and in the most preferred embodiment at least 3 different pathways are represented by the frameshift polypeptides in a set according to the present invention.
  • the TGF ⁇ RII as a member of a signal transduction pathway may be used in combination with the BAX gene as a member of the apoptosis pathway with additional other polypeptides associated with other pathways.
  • a final aspect influencing the choice of suitable frameshift polypeptides to be included in the set according to the present invention is the length of the novel (poly)peptide sequence arising from the mutation.
  • the shift of the reading frame leads to novel stop codons.
  • the new peptides are not of the same length as the polypeptides naturally encoded by the particular gene. In most cases the new peptides are shorter or even significantly shorter than the original wild-type polypeptide. Frequently rather oligopeptides arise from the frameshift mutations.
  • the fidelity of the immune system in recognizing "foreign" molecules reaches thus far, to identify even polypeptides, that differ from "own” polypeptides in only one single amino-acid mutation.
  • the fragments, bound by the antigen presenting HLA molecules comprise about 12 amino acid residues.
  • more than one single amino-acid difference should be present.
  • a polypeptide comprising 3 consecutive amino acids differing from the wild-type amino acid sequence is reliably recognized as foreign by the immune system. This may be due to the increased probability of new amino acid combinations being present in different fragments produced by the antigen presenting machinery.
  • the frameshift polypeptides according to the present invention contain at least one new amino acid, not present in the wild-type polypeptide, in a more preferred embodiment at least 2 new amino acids and in the most preferred embodiment at least 3 new amino acids.
  • a basic set of frameshift polypeptides may be tailored, that is suitable to address a large variety of tumours and minimizes the danger of escape of single tumour cells from the therapy.
  • the probability of survival of tumour cells in an organism following immuno-therapy can be minimized and the rate of recurrence of the cancer can be reduced.
  • a basic set of frameshift polypeptides includes frameshift polypeptides, that do occur with a high mutation frequency in associated disorders. Additionally the polypeptides within the set are chosen to be involved in different biochemical pathways. The mutation types are chosen, that polypeptides of a minimal length of 3 amino acid residues is expressed from the mutated nucleic acid sequence. Furthermore different mutation types of one single microsatellite may be included in the set if applicable.
  • compositions and methods according to the present invention may be applied to any eukaryotic organisms exhibiting an immunologic defense system.
  • the eukaryotic organisms are for example animals of agricultural value such as pigs, cows, sheep, etc., companion animals, such as cats, dogs, horses etc., animals employed in research purposes such as mice, rats, rabbits, hamsters etc. or humans.
  • frameshift polypeptides that comprise an immunogenic portion may be used for immunotherapy for the treatment of cancer.
  • Immunotherapy may be broadly classified into either active or passive immunotherapy.
  • active immunotherapy treatment relies on the in vivo stimulation of the endogenous host immune system to react against tumours with the administration of immune response-modifying agents (for example, tumour vaccines, bacterial adjuvants, and/or cytokines).
  • immune response-modifying agents for example, tumour vaccines, bacterial adjuvants, and/or cytokines.
  • a patient may be afflicted with disease, or may be free of detectable disease.
  • the compounds disclosed herein may be used to treat cancer or to inhibit the development of cancer.
  • the compounds are preferably administered either prior to or following primary treatment of tumours such as surgical removal of the tumours, treatment by administration of radiotherapy and/or conventional chemotherapeutic drugs or any other mode of treatment of the respective cancer or its precursors.
  • tumour-immune reactivity such as effector cells or antibodies
  • effector cells include T lymphocytes (for example, CD8+ cytotoxic T-lymphocytes, CD4+ T-helper, tumour-infiltrating lymphocytes), killer cells (such as Natural Killer cells, lymphokine-activated killer cells), B cells, or antigen presenting cells (such as dendritic cells and macrophages) expressing the disclosed antigens.
  • T lymphocytes for example, CD8+ cytotoxic T-lymphocytes, CD4+ T-helper, tumour-infiltrating lymphocytes
  • killer cells such as Natural Killer cells, lymphokine-activated killer cells
  • B cells or antigen presenting cells (such as dendritic cells and macrophages) expressing the disclosed antigens.
  • antigen presenting cells such as dendritic cells and macrophages
  • the predominant method of procuring adequate numbers of T-cells for adoptive transfer immunotherapy is to grow immune T-cells in vitro.
  • Culture conditions for expanding single antigen-specific T-cells to several billion in number with retention of antigen recognition in vivo are well known in the art.
  • These in vitro culture conditions typically utilize intermittent stimulation with antigen, often in the presence of cytokines, such as IL-2, and non-dividing feeder cells.
  • cytokines such as IL-2
  • the immunoreactive polypeptides described herein may be used to rapidly expand antigen-specific T-cell cultures in order to generate sufficient number of cells for immunotherapy.
  • antigen-presenting cells such as dendritic-, macrophage- or B-cells
  • antigen presenting cells may be transfected with a nucleic acid sequence, wherein said sequence contains a promoter region appropriate for increasing expression, and can be expressed as part of a recombinant virus or other expression system.
  • the cultured T-cells must be able to grow and distribute widely and to survive long term in vivo.
  • sets of frameshift polypeptides may be employed to generate and/or isolate tumour-reactive T-cells, which can then be administered to the patient.
  • antigen-specific T-cell lines may be generated by in vivo immunization with short peptides corresponding to immunogenic portions of the disclosed polypeptides.
  • the resulting antigen specific CD8+ CTL or CD4+ T-helper cells clones may be isolated from the patient, expanded using standard tissue culture techniques, and returned to the patient.
  • peptides corresponding to immunogenic portions of the polypeptides of the invention may be employed to generate tumour reactive T-cell subsets by selective in vitro stimulation and expansion of autologous T-cells to provide antigen-specific T-cells which may be subsequently transferred to the patient as described, for example, by Chang et al. (Crit. Rev. Oncol. Hematol., 22(3), 213, 1996).
  • Cells of the immune system such as T-cells, may be isolated from the peripheral blood of a patient, using a commercially available cell separation system, such as CellPro Incorporated's (Bothell, Wash.) CEPRATE.TM. system (see U.S. Pat. No. 5,240,856; U.S. Pat. No.
  • the separated cells are stimulated with one or more of the immunoreactive polypeptides contained within a delivery vehicle, such as a microsphere, to provide antigen-specific T-cells.
  • a delivery vehicle such as a microsphere
  • the population of tumour antigen-specific T-cells is then expanded using standard techniques and the cells are administered back to the patient.
  • T-cell receptors and/or antibodies specific for the polypeptides can be cloned, expanded, and transferred into other vectors or effector cells for use in adoptive immunotherapy.
  • syngeneic or autologous dendritic cells may be pulsed with peptides corresponding to at least an immunogenic portion of a polypeptide disclosed herein.
  • the resulting antigen-expressing and/or presnting dendritic cells may either be transferred into a patient, or employed to stimulate T-cells to provide antigen-specific T-cells which may, in turn, be administered to a patient.
  • the use of peptide-pulsed dendritic cells to generate antigen-specific T-cells and the subsequent use of such antigen-specific T-cells to eradicate tumours in a murine model has been demonstrated by Cheever et al, (Immunological Reviews, 157:177, 1997).
  • Monoclonal antibodies directed against frameshift peptides presented on cellular membranes may according to the present invention also be used as therapeutic compounds in order to diminish or eliminate tumours.
  • the antibodies may be used on their own (for instance, to inhibit metastases) or coupled to one or more therapeutic agents.
  • Suitable agents in this regard include radionuclides, differentiation inducers, drugs, toxins, and derivatives thereof.
  • Preferred radionuclides include 90Y, 1231, 1251, 1311, 186Re, 188Re, 211At, and 212Bi.
  • Preferred drugs include methotrexate, and pyrimidine and purine analogs.
  • Preferred differentiation inducers include phorbol esters and butyric acid.
  • Preferred toxins include ricin, abrin, diptheria toxin, cholera toxin, gelonin, Pseudomonas exotoxin, Shigella toxin, and pokeweed antiviral protein.
  • compositions useful in immuno-therapy may comprise a set of at least 5 to 10 frameshift polypeptides (or variants thereof), and a physiologically acceptable carrier.
  • the vaccines may additionally comprise a non-specific immuneresponse enhancer, wherein the non-specific immune response enhancer is capable of eliciting or enhancing an immune response to an exogenous antigen.
  • non-specific-immune response enhancers include adjuvants, biodegradable microspheres (e.g., polylactic galactide) and liposomes (into which the polypeptide is incorporated).
  • Pharmaceutical compositions and vaccines may also contain other epitopes of tumour antigens, either incorporated into a fusion protein or present within a separate polypeptide.
  • a pharmaceutical composition or vaccine suitable for immunotherapy may contain nucleic acids, that code for one or more frameshift polypeptides according to the present invention.
  • Nucleic acids may for example include single-stranded (sense or antisense) or double-stranded molecules, and may be DNA (genomic, cDNA or synthetic) or RNA.
  • RNA molecules comprise as well HnRNA (containing introns) as mRNA (not containing introns).
  • the polynucleotides may also be linked to any other molecules, such as support materials or detection marker molecules, and may, but need not, contain additional coding or non-coding sequences.
  • the nucleic acid may be administered in a way that allows the polypeptides to be generated in situ.
  • Suitable expression systems are known to those skilled in the art.
  • the expression of the polypeptides may for example be persistent or transient.
  • the nucleic acids may be present within any suitable delivery system known to those of ordinary skill in the art, including nucleic acid expression systems, bacteria and viral expression systems.
  • Appropriate nucleic acid expression systems comprise the necessary regulatory nucleic acid sequences for expression in the patient, such as suitable promoters, terminators etc..
  • Bacterial delivery systems may for example employ the administration of a bacterium that expresses an epitope of a cell antigen on its cell surface.
  • the nucleic acid may be introduced using a viral expression system such as e.g., vaccinia virus, retrovirus, or adenovirus, which may involve the use of a non-pathogenic, replication competent virus.
  • a viral expression system such as e.g., vaccinia virus, retrovirus, or adenovirus
  • vaccinia virus e.g., vaccinia virus, retrovirus, or adenovirus
  • Suitable systems are known to those of ordinary skill in the art and are disclosed, for example, in Fisher-Hoch et al., PNAS 86:317-321, 1989; Flexner et al., Ann. N.Y. Acad Sci. 569:86-103, 1989; Flexner et al., Vaccine 8:17-21, 1990; U.S. Pat. Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89/01973; U.S.
  • transgenic mammalian cells may be used for delivery and/or expression of the nucleic acids.
  • the methods for producing nucleic acid constructs suitable for in-situ expression of polypeptides are known to those of skill in the art.
  • nucleic acid may be administered as naked nucleic acids.
  • appropriate physical delivery systems which enhance the uptake of nucleic acid may be employed, such as coating the nucleic acid onto biodegradable beads, which are efficiently transported into the cells.
  • Administration of naked nucleic acids may for example be useful for the purpose of transient expression within a host or host cell.
  • compositions used for immuno-therapy according to the present invention may be administered by any suitable way known to those of skill in the art.
  • the administration may for example comprise injection, such as e.g., intracutaneous, intramuscular, intravenous or subcutaneous injection, intranasal administration for example by aspiration or oral administration.
  • a suitable dosage to ensure the pharmaceutical benefit of the treatment should be chosen according to the parameters, such as age, sex, body weight etc. of the patient, known to those of skill in the art.
  • the carrier preferably comprises water, saline, alcohol, a lipid, a wax and/or a buffer.
  • the carrier preferably comprises water, saline, alcohol, a lipid, a wax and/or a buffer.
  • any of the above carriers or a solid carrier such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, and/or magnesium carbonate, may be employed.
  • Biodegradable microspheres e.g., polylactic glycolide
  • suitable biodegradable microspheres are disclosed, for example, in U.S. Pat. Nos.
  • any of a variety of immune-response enhancers may be employed in the vaccines of this invention.
  • an adjuvant may be included.
  • Most adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a nonspecific stimulator of immune response, such as lipid A, Bordetella pertussis or Mycobacterium tuberculosis.
  • lipid A lipid A
  • Bordetella pertussis or Mycobacterium tuberculosis lipid A
  • Such adjuvants are commercially available as, for example, Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.) and Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.).
  • compositions or immuno-therapeutic methods according to the present invention may be used for the treatment of degenerative disorders or cancers.
  • the compositions and methods may be employed in the therapy of diagnosed cancers in order to eliminate the tumour cells from the affected organism.
  • primary tumours as metastases or disseminated tumour cells within an organism may be targets to the therapeutic compounds and methods disclosed herein.
  • compositions and methods of the invention may be employed in the treatment of pre-neoplastic conditions.
  • the pre-neoplastic cells or tissues may be directly addressed by the immuno-therapeutic compositions or methods, or may be hindered from evolving into neoplastic or dysplastic conditions.
  • the pre-neoplastic condition may be treated preventively.
  • the immune response may be elicited, so that emerging neoplasms may be destroyed.
  • the methods and compositions according to the present invention may also be used for the prevention of degenerative disorders or cancers associated with frameshift mutations in coding microsatellites.
  • a vaccination of a population of organisms or of subgroups of said population may be performed.
  • the subgroups may be built by suitable parameters such as hereditary predisposition for the emergence of degenerative disorders, exposure to factors, that increase the risk of being affected by said disorders etc.
  • the present invention provides compositions and methods for enhanced immunotherapy of disorders associated with MSI + related occurrence of frameshift peptides.
  • the invention provides sets for the immuno-therapy of said disorders, that address a wide range of different types of disorders in an organism and may thus be employed as a preventive vaccine against said disorders.
  • the invention also provides sets of polypeptides useful for the treatment of particular types of disorders.
  • the use of sets of frameshift polypeptides for the immuno-therapy according to the present invention provides the means for a reliable treatment of degenerative disorders and cancers associated with frameshift mutations in coding microsatellites reducing the risk of escape of several tumour cells or of a population of tumour cells from being addressed by the therapy.
  • the method of the invention reduces the risk of recurrence of tumours after immuno-therapy employing CTLs, T-helper cells and possibly specific antibody producing B-cells raised against frameshift polypeptides characteristic for tumour cells.
  • Example 1 Analysis of the mutation frequency of genes harbouring repeat tracts including data from publications and own data
  • a minimum repeat length of 6 mononucleotides and 4 dinucleotides was required as lower limit of the repeats.
  • all 12 different types of dinucleotide repeats were taken into consideration.
  • cDNA and genomic DNA entries were analyzed separately. Using several filters, repeat tracts within pseudogenes, vector sequences as well as homopolymeric nucleotide stretches at the most 5' or 3' ends of sequences were excluded. All candidate sequences were stored in a relational database for further analysis.
  • PCR primers were designed to closely flank the target sequence, yielding short amplimeres of about 100 base pairs thus allowing precise fragment sizing and robust amplification from archival tissues.
  • PCR reactions were performed in a total volume of 25 ⁇ l containing 50 ng genomic DNA, 2.5 ⁇ l 10 x reaction buffer (Life Technologies, Düsseldorf, Germany), 1.5 mM MgCl 2 , 200 ⁇ M dNTPs, 0.3 ⁇ M of each primer, and 0.5 U Taq DNA polymerase (Life Technologies) and using the following conditions: initial denaturation at 94 °C for 4 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 58 °C for 45 s, and primer extension at 72 °C for 30 s.
  • the final extension step was carried out at 72 °C for 6 min.
  • PCR fragments were analyzed on an ALF DNA sequencing device (Amersham Pharmacia Biotech, Freiburg, Germany) using 6.6 % polyacrylamide/ 7 M urea gels. Size, height and profile of microsatellite peaks were analyzed using the AlleleLinks software (Amersham Pharmacia Biotech). Coding microsatellite instability was scored, if smaller or larger-sized amplimeres were detected in tumour DNA compared to DNA from non-neoplastic cells. Allele intensities were determined and ratios of wild-type and novel alleles in normal and tumour tissues were calculated, defining a two-fold difference as threshold for allelic shifts.
  • tumour cell lines were identified by comparison to 36 unmatched normal mucosae.
  • Big Dye terminator cycle sequencing Perkin Elmer, Darmstadt, Germany
  • the frequencies of mutations in a particular microsatellite region examined herein are given in figure 1.
  • the mutation rates are calculated with respect to the total number of samples included in the studies.
  • the mutations frequencies resulting from studies covering a larger number of samples may be handled to be more significant then those referring to a smaller number of analysed samples.
  • Example 2 Detection of the expression of frameshift mutated mRNA from genes harboring coding microsatellite regions using PCR
  • Samples of colon, gastric and endometrial carcinomas are used to determine the expression of mRNA showing frameshift mutations in coding microsatellite regions using PCR and subsequent sequencing of the amplified nucleic acid products.
  • Tumours are collected, snap frozen, and stored at -80°C. They are verified to be composed predominantly of neoplastic cells by histopathological analysis. mRNA is isolated from tumours using Qiagen reagents (Qiagen, Hilden, Germany), and single-stranded cDNA is synthesized using Superscript II (Life Technologies, Inc.).
  • PCR reactions were performed in a total volume of 25 ⁇ l containing 50 ng cDNA, 2.5 ⁇ l 10 x reaction buffer (Life Technologies, Düsseldorf, Germany), 1.5 mM MgCl 2 , 200 ⁇ M dNTPs, 0.3 ⁇ M of each primer, and 0.5 U Taq DNA polymerase (Life Technologies) and using the following conditions: initial denaturation at 94 °C for 4 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 58 °C for 45 s, and primer extension at 72 °C for 30 s. The final extension step was carried out at 72 °C for 6 min. PCR fragments were analyzed on an ALF DNA sequencing device (Amersham Pharmacia Biotech, Freiburg, Germany) using 6.6 % polyacrylamide/ 7 M urea gels.
  • the present experiments were performed in order to determine, whether the frameshift peptides arising from mutations in coding microsatellite regions according to the present invention are suited to stimulate a cellular immune response.
  • the experiments were performed as follows:
  • Peptides displaying HLA-A2.1-binding motifs were selected by taking advantage of specific computer programs [(Parker, Bednarek, & Coligan 1994); http://bimas.dcrt.nih.gov/molbio/hla_bind/ and (Rammensee et al. 1999); http://134.2.96.221/scripts/MHCServer.dll/home.htm].
  • Peptides were purchased from the peptide synthesis unit of the DKFZ. Stock solutions (10mg/ml in DMSO) were stored at -70°C and diluted to 1mg/ml in PBS before use.
  • T2 cells were pulsed with 50 ⁇ g/ml peptide and 5 ⁇ g/ml ⁇ 2-microglobulin (Sigma; Deisenhofen, Germany) overnight at 37°C.
  • the expression of HLA-A2.1 was then analysed by flow cytometry using mAb BB7.2 followed by incubation with FITC-conjugated (Fab')2 goat antimouse Ig (Vonderheide et al. 1999).
  • Peripheral blood was obtained from a healthy HLA-A2.1 + donor and collected in heparinized tubes.
  • PBMNC were isolated by Ficoll-density gradient centrifugation.
  • Whole CD3+ T-cells were isolated from PBMNC by magnetic depletion of non-T-cells using the MACS Pan T-Cell Isolation Kit (Miltenyi; Bergisch Gladbach, Germany) according to manufacturer's instructions. Preparations contained at least 97% of CD3+ cells as assessed by immunophenotypic analysis.
  • CD40 Bs of a HLA-A2.1+ donor were incubated with peptide (10 ⁇ g/ml) and human ⁇ 2-microglobulin (3 ⁇ g/ml; Sigma) in serum-free Iscov's DMEM medium for one hour at room temperature, washed twice to remove excess of peptide, were irradiated (30Gy) and added to purified CD3+ autologous T-cells (>97% CD3+) at a ratio of 4:1 (T:CD40 Bs) in Iscov's MEM containing 10% human AB-serum, supplements (1:100) and hIL-7 (10ng/ml, R&D).
  • IL-2 was first given at day 21 (10IU/ml, R&D), also at day 24 and from day 28 on only hIL-2 was used instead of hIL7.
  • ELISpot assays were performed as described elsewhere (Meyer et al. 1998). Briefly, nitrocellulose-96-well plates (Multiscreen; Millipore, Bedford, USA) were covered with mouse anti-human IFN-g monoclonal antibody (Mabtech, Sweden) and blocked with serum containing medium. Varying numbers of effector cells were plated in triplicates with 3,5x10 4 peptide-loaded T2 cells per well as targets. After incubation for 18h, plates were washed, incubated with biotinylated rabbit anti-human IFN-g second antibody, washed again, incubated with streptavidin coupled alkaline phosphatase, followed by a final wash. Spots were detected by incubation with NBT/BCIP (Sigma) for 45min, reaction was stopped with water, after drying spots were counted using the KS-ELISpot reader (Zeiss Kontron; Göttingen, Germany).
  • Example 4 Vaccination therapy using a set of 10 preselected frameshift polypeptides
  • a set of 10 frameshift polypeptides preselected by rational considerations is used for carrying out the immunotherapy.
  • the set of polypeptides consisted of frameshift peptides derived from mutations in the coding regions of the following genes: TGF ⁇ RII, U79260, CASP 5, HT001, PTHL3, MACS, TCF4, TAF1B, AC1 and SEC63
  • the peptide-mixture used for vaccination consists of equimolar amounts of the different (10) freeze-dried peptides.
  • the peptides are dissolved in 0.5ml sterile 0.9% NaCl at a final concentration of 1-1.6mg/ml for each peptide.
  • Vaccination is carried out using 100 ⁇ l peptide mixture (5 x 10 -5 M concentration of each peptide). The mixture is administered intracutaneously about 20min after injection of 100 ⁇ l (0.4mg/ml) recombinant human GM-CSF (i.e. Leucomax®, Schering-Plough). 100 ⁇ l of peptide mixture will be injected intracutaneously at a separate site without GM-CSF as a DTH-test. A DTH-test with each individual peptide is performed at week 6 to distinguish between positive and negative reactions to the individual peptides. The DTH-test is considered positive if the area of the skin reaction has a diameter of at least 5mm.
  • Vaccination regimen includes 4 vaccinations carried out weekly. The first vaccination is performed a minimum of four weeks after other treatments have been stopped.
  • the assessment of the tumor response is carried out by CT or X-Ray examination as applicable in the particular case.
  • the assessment is performed two months prior to the first vaccination and two months after the last vaccination.
  • tumor entities to which the presented set will be applicable include breast cancer, cancer of the brain, endometrial cancer and others.

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EP02008774A EP1369126A1 (fr) 2002-04-18 2002-04-18 Utilisation de peptides de régions de microsatellites codantes et mutantes de déphasage pour traiter le cancer
PCT/EP2003/004083 WO2003087162A2 (fr) 2002-04-18 2003-04-17 Neopeptides et procedes utilises dans la detection et le traitement du cancer
AU2003232485A AU2003232485A1 (en) 2002-04-18 2003-04-17 Neopeptides and methods useful for detection and treatment of cancer
US10/511,698 US8053552B2 (en) 2002-04-18 2003-04-17 Neopeptides and methods useful for detection and treatment of cancer
US13/284,660 US8821864B2 (en) 2002-04-18 2011-10-28 Compounds and methods useful for detection and treatment of cancer
US14/340,467 US20140363831A1 (en) 2002-04-18 2014-07-24 Compounds and methods useful for detection and treatment of cancer
US16/120,981 US20190265244A1 (en) 2002-04-18 2018-09-04 Compounds and Methods Useful for Detection and Treatment of Cancer
US17/302,694 US20220091122A1 (en) 2002-04-18 2021-05-10 Compounds and Methods Useful for Detection and Treatment of Cancer

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Cited By (8)

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EP1994181A2 (fr) * 2006-02-27 2008-11-26 Arizona Board of Regents, acting for and on behalf of Arizona State University Identification et utilisation de novopeptides pour le traitement du cancer
KR100998272B1 (ko) * 2002-07-15 2010-12-08 연세대학교 산학협력단 단백질 지령부위내 단반복 염기서열을 갖는 신규 유전자
US9732131B2 (en) 2006-02-27 2017-08-15 Calviri, Inc. Identification and use of novopeptides for the treatment of cancer
WO2018223093A1 (fr) * 2017-06-02 2018-12-06 Arizona Board Of Regents On Behalf Of Arizona State University Vaccins anticancereux universels et leurs procédés de préparation et d'utilisation
WO2021142007A1 (fr) * 2020-01-09 2021-07-15 Arizona Board Of Regents On Behalf Of Arizona State University Anticorps et procédé de production d'anticorps
US11484581B2 (en) 2017-06-02 2022-11-01 Arizona Board Of Regents On Behalf Of Arizona State University Method to create personalized canine cancer vaccines
US11971410B2 (en) 2017-09-15 2024-04-30 Arizona Board Of Regents On Behalf Of Arizona State University Methods of classifying response to immunotherapy for cancer
US11976274B2 (en) 2019-10-02 2024-05-07 Arizona Board Of Regents On Behalf Of Arizona State University Methods and compositions for identifying neoantigens for use in treating and preventing cancer

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100998272B1 (ko) * 2002-07-15 2010-12-08 연세대학교 산학협력단 단백질 지령부위내 단반복 염기서열을 갖는 신규 유전자
EP1994181A2 (fr) * 2006-02-27 2008-11-26 Arizona Board of Regents, acting for and on behalf of Arizona State University Identification et utilisation de novopeptides pour le traitement du cancer
EP1994181A4 (fr) * 2006-02-27 2010-05-19 Univ Arizona Identification et utilisation de novopeptides pour le traitement du cancer
US9732131B2 (en) 2006-02-27 2017-08-15 Calviri, Inc. Identification and use of novopeptides for the treatment of cancer
US11168121B2 (en) 2006-02-27 2021-11-09 Calviri, Inc. Identification and use of novopeptides for the treatment of cancer
EP3630152A4 (fr) * 2017-06-02 2021-07-28 Arizona Board of Regents on behalf of Arizona State University Vaccins anticancereux universels et leurs procédés de préparation et d'utilisation
WO2018223093A1 (fr) * 2017-06-02 2018-12-06 Arizona Board Of Regents On Behalf Of Arizona State University Vaccins anticancereux universels et leurs procédés de préparation et d'utilisation
US11484581B2 (en) 2017-06-02 2022-11-01 Arizona Board Of Regents On Behalf Of Arizona State University Method to create personalized canine cancer vaccines
US11971410B2 (en) 2017-09-15 2024-04-30 Arizona Board Of Regents On Behalf Of Arizona State University Methods of classifying response to immunotherapy for cancer
US12025615B2 (en) 2017-09-15 2024-07-02 Arizona Board Of Regents On Behalf Of Arizona State University Methods of classifying response to immunotherapy for cancer
US11976274B2 (en) 2019-10-02 2024-05-07 Arizona Board Of Regents On Behalf Of Arizona State University Methods and compositions for identifying neoantigens for use in treating and preventing cancer
US12018252B2 (en) 2019-10-02 2024-06-25 Arizona Board Of Regents On Behalf Of Arizona State University Methods and compositions for identifying neoantigens for use in treating cancer
WO2021142007A1 (fr) * 2020-01-09 2021-07-15 Arizona Board Of Regents On Behalf Of Arizona State University Anticorps et procédé de production d'anticorps

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